1. Field of the Invention
The present invention relates to a method for cell-free protein synthesis. More specifically, the present invention relates to a synthesis method capable of improving an amount of synthesized protein per unit time with a simple method.
2. Disclosure of the Related Art
In recent years, genetic information of many organisms, such as human genome, has been decoded. Under the circumstances, functional analysis of proteins and creation of genomic medicine based on such genetic information have been attracting attention for postgenomic studies. Application and utilization of proteins corresponding to such genetic information for pharmaceutical products and the like requires easy synthesis of extensive kinds of proteins in a short time.
At present, expression systems using viable cells (hereinafter sometimes to be referred to as “cell-system”) of yeast, insect cell and the like by the gene recombination technique have been widely utilized as the production methods of proteins. However, viable cells show a propensity toward elimination of exogenous proteins for their functional retention, and there are many proteins that cannot be expressed easily since expression of cytotoxic proteins in viable cells prevents cell growth.
On the other hand, as a production method of protein free of a cell-system, cell-free protein synthesis has been known, which includes adding a substrate, an enzyme and the like to a cell rupture, extract solution and the like to provide a wide choice of genetic information translation systems of organisms in test tubes, and reconstructing a synthetic system capable of linking the necessary number of amino acid residues in a desired order using an mRNA encoding an object protein. Such a cell-free protein synthesis is relatively free of the limitation imposed on the above-mentioned cell-system protein synthesis, and is capable of synthesizing proteins without killing the organism. In addition, because the production of protein does not accompany operations of culture and the like, the protein can be synthesized in a short time as compared to cell-systems. Moreover, inasmuch as the cell-free protein synthesis also affords a large scale production of proteins consisting of amino acid sequences not utilized by the organism, it is expected to be a promising expression method. However, a cell-free protein synthesis system has a problem in that reaction time is shorter and the amount of a synthesized protein is extremely smaller as compared to a cell-system. In order to overcome such a problem of a batch method which is a conventional cell-free protein synthesis method, various studies have been carried out.
For example, there has been reported that a protein can be continuously synthesized by a method of feeding a reaction solution to an ultrafilter (flow method) in A. S. Spirin et al., Science, 242, 1162-1164 (1988) or JP-B-07-110236. However, such a flow method requires large-scale devices, and is therefore disadvantageous in terms of cost.
JP-A-2002-539840 describes a cell-free protein synthesis method by a method of using a dialyzer (dialysis method). In this method, a wheat germ extract is used to synthesize CAT by means of a translation system or a coupled transcription/translation system. Further, Mg+ and NTP concentrations are continuously changed during synthesis to improve the yield of CAT. However, in the case of a translation system, the yield of CAT obtained by this method is about 17 μg/mL. On the other hand, in the case of a coupled transcription/translation system in which Mg+ and NTP concentrations are continuously changed, the yield of CAT obtained by this method is 32 μg/mL at best.
Further, on the other hand, as a cell rupture or extract solution to be applied to the cell-free protein synthesis, use of various substances of biological derivation has been considered and investigations are underway. Of such substances, since insect cells do not require, unlike many mammalian culture cells, culture under a carbon dioxide atmosphere, can be cultured in a serum-free medium, and can express in a large amount in a cell-system while retaining the inherent biological activity with posttranslational modification, they are used for the expression of various proteins. If the insect cell can be used for a cell-free system, posttranslational modification, such as glycosylation and the like, is fully expected to be applicable. Thus, the development of utilization of the insect cell is drawing attention.
As a method for preparing an extract solution for cell-free protein synthesis, a method of extraction from an insect cell has been disclosed in JP-A-2000-325076, JP-A-2004-215651, or the like. JP-A-2000-325076 discloses a method for reducing a pressure applied to an insect cell after pressurization in an inert gas atmosphere, thereby to rupture the insect cell to allow for extraction. JP-A-2004-215651/discloses, as a method for easily preparing an insect cell extract solution which affords synthesis of a large amount of protein, a method for preparing an insect cell extraction solution for cell-free protein synthesis, the method comprising at least a step of rapidly freezing an insect cell suspended in a solution for extraction. In addition, JP-A-2004-215651 also discloses a method for cell-free protein synthesis in accordance with a batch method, using the insect cell extract solution prepared as described above.